Water soluble luminescent nanoparticles

ABSTRACT

A particle of a compound of the formula: X a (YO b ) c  wherein X is a rare earth metal or a metal of Group IIA, IIB, IVB or VB of the Periodic Table, or a mixture of two or more thereof, Y is a metal which forms an anion with oxygen, or a mixture of two or more thereof, and a, b and c are such that the compound is stoichiometric, the particle having a size less than 100 nm is disclosed. It can be used in, for example, security marking and biotagging.

This invention relates to the preparation of water-soluble quantum dotsor nanoparticles which are particularly useful in biological tagging andsecurity tagging.

The use of common organic dyes for tagging presents many problems, inparticular due to photobleaching and because the narrow absorption bandsmake it difficult to excite different colours at once. Dye emission canalso be broad, making multicolour imaging difficult. Previous attemptsto utilise luminescent quantum dots for tagging applications have morerecently been based principally on semiconductors, with luminescence ofvarious colours being generated by transitions across the quantumconfined semiconductor band gap. The size of the nanoparticles governsthe wavelength of the emission. This approach has a number ofsignificant drawbacks:

(i) Semiconductors with suitable bulk band gaps are based on materialssuch as group III/V or group II/VI materials. Typically, CdSe or CdS areused. These materials are toxic, and synthesis is generally carried outin organic solvents. Therefore, phase transfer to water is requiredafter they have been prepared. This is technologically difficult tocarry out while maintaining luminescence efficiency. Quantum dots whichcan be formed in water remove a significant barrier to synthesis.

(ii) If semiconductors are used then size selection must be used toseparate material of different emission wavelengths. This leads to asubstantial loss of material for a single synthesis run while requiringan additional step which involves the use of specialist equipment.

(iii) Typical semiconductor materials are toxic, and their precursorsmay be highly toxic. Also they are frequently air/moisture sensitive.

(iv) To make highly luminescent particles requires a further shell ofsemiconductor of a wider bandgap and often a further shell of silica.

There is therefore a need for water-soluble quantum dot materials(generally ≦100 nm) which are non-toxic and which can be preparedefficiently without the need for specialist apparatus.

It has been found that these needs can be met by certain phosphors whichhave the advantage that they are intrinsically luminescent, non-toxicand can be synthesised by a route which requires no specialistapparatus.

According to the present invention there is provided a particle of acompound of the formula:X_(a)(YO_(b))_(c)

-   -   wherein X is a rare earth metal or a metal of group II A, IIB,        IVB or VB the Periodic Table, or a mixture of two or more        thereof, Y is a metal which forms an anion with oxygen, or a        mixture of two or more thereof, and a, b and c are such that the        compound is stoichiometric, the particle having a size less than        100 nm.

The use of rare earths in this way is particularly surprising since itis known that they are susceptible to concentration quenching i.e. astheir concentration increases their emission gets quenched by adjacentrare earth ions.

Typical metals for X include rare earth metals such as Eu, Dy, Th, Ce,Sm, Er, Th, Gd and Pr, as well as Yb and Ho. Suitable metals of group IIA and B include magnesium, calcium and zinc while metals of groups IVBand VB include bismuth tin and lead. In one embodiment the group IIAmetal is not calcium and the compound is not CaWO₄.

The metals for Y are those which form an anion with oxygen so that theyare in the form of metalates. Preferred metals for Y include tungsten,vanadium, molybdenum, niobium and tantalum.

The simplest compounds are generally those where a is I, b is I and c is4 as in Eu(MoO₄) but other phases of such compounds exist, for exampleEu₂(MoO₄)₃. The compounds can have more than two metals present. Thusthe compound may be derived from more than one metal X and/or more thanone metal Y. The use of mixtures such as mixed anions provides acombination of active centres excitable to optimise absorptioncharacteristics beyond those obtainable for each anion independently. Aspecific example is where Y is a mixture of vanadium and tungsten. Suchcompounds typically respond to excitation wave lengths of the order of320 nm. Mixed cations can be used to produce compounds where thedistribution of luminescent ions is modified such that concentrationquenching effects, for example, are reduced/minimised. A specificexample is where X is a mixture of Gd and Eu. Thus the compound may be amixed vanadate/tungstate salt of gadolinium and europium.

The particles of the present invention are quantum dots having aparticle size not exceeding or less than 100 nm, typically not exceeding50 nm, for example 1 to 50 nm. The particles can be formed withoutdifficulty with a particle size less than or not exceeding 10 nm, forexample 2 or 3 nm to 10 nm.

The particles of the present invention can be prepared by a processwhich comprises mixing an aqueous solution having a basic pH of acompound containing an anion of Y and a surfactant with is an organicacid or a Lewis base, with an aqueous solution of a compound containingthe cation X.

Thus a water-soluble compound containing an anion of Y can be added towater, generally with stirring, and a suitable surfactant is added toit. The surfactant has the effect of passivating the surface, stoppingparticle growth and maintaining luminescence efficiency. It will beappreciated that particle growth will generally give rise tosubstantially crystalline particles. The pH is then generally increased,generally by addition of a base. The purpose of this is to maintain thecorrect anion/cation ratio in the precipitated materials. In general,the pH should be maintained at least 8 and typically 8 to 10, forexample 8 to 9.

The material can then be precipitated by the addition of a solution of awater-soluble compound containing the cation, generally with stirring.Usually, the quantum dot material forms instantly and luminescence isclearly visible.

The water-soluble compound containing an anion of Y is typically analkali metal salt such as a sodium salt e.g. sodium tungstate althoughammonium salts such as 5(NH₄)₂O. 12WO₃5H₂O can also be used.

Surfactants which can be used are organic acids and Lewis bases whichare generally polar.

For the ligand/surface active molecule to be effective it must be ableto stick to the particle surface. Typically compounds which can achievethis include phosphines, phosphine oxides, thiols, amines, carboxylicacids, phosphates, such as sodium hexametaphosphate, which is preferred,sulfonic acids, sulfinic acids, phosphoric acids, phosphonic acids,phosphinic acids, crown ethers and mixtures of these. The compound usedwill, of course, depend on the nature of the particle as one skilled inthe art will appreciate. For example it is believed that cerium attractscarboxylic acid groups. However tungstates and the like generallyattract phosphate groups.

The ligand itself can be monodentate (i.e. with a single binding point,e.g. a trialkylphosphine oxide e.g. with a chain length of 4 to 20carbon atoms), bidentate (e.g. dihydrolipoic or a dialkylsulphosuccinate e.g. sodium dioctyl sulphosuccinate with a similar chainlength to monodentate) or multi dentate (polymer/dendrimers with pendantside groups such as phosphines, phosphine oxides, thiols, amines,carboxylic acids, phosphates, sulfonic acids, sulfinic acids, phosphoricacids, phosphinic acids and mixtures of).

The ligand can also be polymeric such as vinyl pyrrolidone or a polymerpossessing, preferably, a carboxylic acid and/or phosphonate group suchas polymers derived from, for example, a vinyl carboxylic acid such asacrylic acid and/or a vinyl monomer possessing a group capable ofbinding to the particles such as vinyl phosphonic acid e.g. Albritech 30which is a copolymer of acrylic acid and vinyl phosphonate.

The ligand should be water soluble. If necessary, therefore, themolecule may contain other groups which assist solubility such ashydroxy and deprotonated acid or protonated amine groups. Thus if apolymer is used it may have side chains that make the ligand watersoluble, e.g. hydroxy groups, deprotonated acids or protonated amines.

Other water-soluble ligands which can be used include sugar molecules,including oligosaccharides, monosaccharides, and polysaccharides whichare water-soluble and contain side groups for further biocouplingreactions such as hydroxy groups as well as amine phosphates, typicallynucleoside phosphates such as adenosine and guanosine phosphatesincluding ATP (adenosine 5′-triphosphate), ADP (adenosine diphosphate),AMP (adenosine monophosphate) and GMP (guanosine monophosphate).Cyclodextrins (cyclic oligosaccharides), functionalised with phosphines,phosphine oxides, thiols, amines, carboxylic acids, phosphates, sulfonicacids, sulfinic acids, phosphoric acids, phosphinic acids and mixturesof can also be used.

It is known that certain metals bind well to certain groups. Accordinglya molecule containing such a group will bind to that metal via thisgroup, leaving the other group (or groups) free for a biocouplingreaction. Thus in many cases a thiocarboxylic acid will coat theparticle with the carboxylic grouping on the surface as the thiol grouphas a stronger affinity for the metal(s) in the particle. Chemical andspectroscopic tests can be made, if necessary, to determine how thecapping agent is oriented.

Generally, the reactants should be used in approximately stoichiometricamounts using a roughly equimolar amount of surfactant and thewater-soluble compound containing Y, although, in general, the relativemolar amounts are from 0.3 to 2. Preferably the molar ratio ofsurfactant to salt is about 1:1 to 2:1. Provided sufficient surfactantis present it can help redispersion of the surfactant once the particleshave been formed. The concentrations of the ingredients in the aqueoussolutions are not particularly critical but generally do not exceedabout 0.1M as if the concentration is to 0 high flocculation of theparticles may occur. Typical concentrations are from 0.005 to 0.1M, i.e.0.01 to 0.05M such as about 0.2M.

Suitable materials which can be used to adjust the pH include alkalissuch as sodium hydroxide, potassium hydroxide and ammonium hydroxide.

The ions of X are introduced as a water-soluble salt of X, preferably ahalide and, in particular, a chloride.

The particles can be obtained as a powder by drying the precipitatewhich is formed, for example in a rotary evaporator. Alternatively, theparticles can be precipitated with a non-aqueous solvent, which ismiscible with water. Suitable such solvents include polar organicsolvents such as aliphatic or aromatic alcohols, especially aliphaticalcohols having 2 to 6, for example 3 or 4, carbon atoms such aspropanol. Other suitable solvents include ethers and light petroleum. Anon-polar solvent can be used with the polar solvent such as analiphatic ketone e.g. acetone. A mixture of propanol and acetone can besuitable. A powder can be obtained by, for example, centrifuging.

The process can generally be carried out at room temperature, andtypically at 0° to 40° C., for example about 20° C. The use of elevatedtemperature tends to result in the luminescence of the particlesdecreasing on standing; this may well be associated with the fact thatas the temperature rises, the surfactant has lower binding strength.

The process can readily be carried out in air. In other words no specialconditions are needed in this respect.

The particles of the present invention find particular utility in thefields of security marking and biological tagging.

For security marking, the quantum dot material is typically formed intoan ink which may be either aqueous or non-aqueous. If they are aqueousthen it is necessary for the surfactant to provide hydrophilic groups onthe surface of the coating. These include —OH, —COOH and —N⁺ (amino oramido) groups. Typical ink formulations involve a binder. Suitablebinders include polymers and resins such as carboxylated acrylic resinsand ethylene/vinylester copolymers e.g. ethylene/vinylacetate copolymerse.g. containing about 40% vinylacetate by weight. Such inks can be usedto print a luminescent security feature on any document or object. It isa particular feature of the particles of the present invention thattheir small size alters the emission profile of the luminescent centrefrom that of bulk material such that unique optical spectra areproduced. This is a particular security feature since it makes it muchmore difficult for the counterfeiter to establish what the luminescentmaterial is.

If the particles are to be used for biological tagging it is necessarythat the particles present a reactive grouping on their surface which iscapable of coupling with a suitable biological molecule. Typical surfacegroups which can be used for this purpose include —SH, —COOH and —N⁺(amino or amido) as well as hydroxy groups. These groups may be atterminal points in the molecule, or as a side chain, and there can bemore than one. These groups can be provided by selecting a surfactantwhich is capable of binding to the surface of the particles while at thesame time providing the appropriate reactive group on the surface. Inthis connection reference should be made to our British application No.0126283.1 (N83808). This application describes a process for preparingwater soluble particles of a luminescent material which is a rare earthmaterial, a doped compound semi-conductor or a doped inorganic compoundwhich comprises coating particles of said luminescent material, eitherduring production of the particles, or subsequently, with an organicacid or Lewis base such that the surface of the coating possesses one ormore reactive groups.

In order to bind the particle to the moiety to be tagged use is made ofa binding interaction between the moiety and a molecule attached to theparticle involving a ligated binding pair. Typically such an interactionis a high affinity non-covalent coupling interaction between a moietyand a molecule able to bind to each other in physiological and/orcellular conditions. The binding may be reversible or non-reversiblebinding.

In one embodiment the moiety itself is the substance which it is desiredto tag, and in this case the moiety will be in a non-modified form, i.e.in its naturally occurring form. In other embodiments the moiety isattached to the substance which it is desired to tag.

One or both of the moiety and molecule on the particle may be a proteinor polynucleotide. Typically one or both of the moiety and molecule arenaturally occurring substances, such as substances found in livingorganisms, for example prokaryotes and/or eukaryotes. In one embodimentthe moiety and molecule are substances which may bind each other whenpresent in their natural locations, such as a receptor ligand pair.

A wide range of moieties can be tagged in this way, for example anycellular component, for example membrane-bound, in the cytoplasm, eitherextra-cellular or intra-cellular. Moieties which move from one cellularlocation to another are particularly useful. The moieties can be presentwithin an organelle, for example in the mitochondria or nucleus. Theyare typically proteins, polynucleotides, carbohydrates or lipids.

Examples of suitable ligand receptor binding pairs include:

-   -   transforming growth factor (TGF) and transforming growth factor        receptor (TGFR) or EGF Receptor (EGFR);    -   epidermal growth factor (EGF) and EGFR;    -   tumor necrosis factor-.alpha. (TNF-.alpha.) and tumor necrosis        factor-receptor (TNFR);    -   interferon and interferon receptor;    -   platelet derived growth factor (PDGF) and PDGF receptor;    -   transferrin and transferrin receptor;    -   avidin and biotin or antibiotin;    -   antibody and antigen pairs,    -   interleukin and interleukin receptor (including types 3, 4 and        5);    -   granulocyte-macrophage colony stimulating factor (GMCSF) and        G,4CSF receptor;    -   macrophage colony stimulating factor (MCSF) and MCSF receptor;        and    -   granulocyte colony stimulating factor (G-CSF) and C—CSF        receptor.

When the moiety is any of the first mentioned substances in the abovepairs then the molecule is generally the second mentioned substance andconversely when the molecule is any of the first mentioned substancesthen the moiety is generally the second mentioned substance. In the caseof the antibody/antigen pair the antigen may be a protein or non-proteinantigen. The antigen may be digoxigenin or phosphotyrosine.

As mentioned above both the molecule and moiety may be polynucleotides.In this case typically the polynucleotides are single stranded and ableto bind to each other by Watson-Crick base pairing, i.e. they arepartially or wholly complementary.

It will be appreciated that the reactive groups on the surface of theparticle are selected such that one member of the pairs will react withthe particle, either directly or with the aid of a crosslinking agent.These are standard reactions well known to those skilled in the art. Forexample, bovine serum albumin can be tagged with amino acid-coatedphosphors using glutaric dialdehyde.

The following Examples further illustrate the present invention.

EXAMPLE 1

Europium Tungstate

100 ml of 0.02M NaWO₄ in water and 100 ml of 0.02M Na(PO₃)₆ surfactantin water are mixed together and stirred for 10 minutes. The pH isadjusted to >8 by dropwise addition of 0.1M NaOH aqueous solution. Afterthe pH adjustment, a 100 ml aqueous solution of 0.02 EuCl₃ is addedunder vigorous stirring. An immediate precipitation is observed; thematerial is quickly redispersed in the water. Under UV illumination thematerial emits bright red luminescence. The excitation and emissionspectra are shown in FIG. 1. The broad excitation peak at 300 nm is dueto transitions to the WO₄ centre and the sharp line at 395 nm is due to4f→4fEu³⁺ transitions. The output consists of two peaks around 600 nm.In bulk Eu³⁺ containing compounds the peak at 611 nm is generallysignificantly stronger than the 590 nm peak. The output spectrum fromthe quantum materials is therefore considerably different.

This unusual output spectrum provides a special feature for securitymarking. In FIG. 2 electron micrographs of typical europium tungstateparticles are shown.

EXAMPLE 2

Terbium Tungstate

100 ml of 0.02M NaWO₄ is added to 100 ml of (NaPO₃)₆ and mixed for 10minutes. The pH is adjusted to >8 by addition of 0.1M NaOH. To this isadded 0.02M TbCl₃. The material is dried and redispersed. The opticalspectrum of the nanoparticles is shown in FIG. 3.

EXAMPLE 3

Europium Molybdenate

100 ml of 0.02M Na₂MoO₄ is added to 100 ml of 0.02M (NaPO₃)₆ and mixedfor 10 minutes. The pH is adjusted to >8 by addition of 0.1M NaOH. Tothis is added 0.02M EuCl₃. The material is dried and redispersed. Theoptical spectrum of the nanoparticles is shown in FIG. 4.

A comparison of FIG. 4 and FIG. 1 shows the effect of substitutingtungstate for molybdenate. The ratio of the two emission peaks haschanged, with the 620 nm peak having a higher intensity relative to the580 nm peak in the molybdenate. In the excitation spectrum the broadabsorption band at 300 nm in the tungstate material has disappeared inthe molybdenate.

EXAMPLE 4

Gd_(13.5)EU_(6.5)(VO₄)_(26.7)(WO₃)_(13.3)This was obtained using the following outline procedure:

-   1) 0.0613 g NaVO₄+0.055 g NaWO₃ are added to 4.5 ml water with 0.5    ml methanol added-   2) 0.186 g GdCl₃ in 5 ml H₂O and 0.214 g EuNO₃ in 5 ml H₂O solutions    are synthesised-   3) 13.5 ml of GdCl₃ soln+6.5 ml of EuNO₃ solution+40 ml of the    VO₄/WO₄ solution forms the material.

1. A particle of a compound of the formula:X_(a)(YO_(b))_(c) wherein x is a rare earth metal or a metal of groupIIA, IIB, IVB or VB of the Periodic Table, or a mixture of two or morethereof, Y is a metal which forms an anion with oxygen, or a mixture oftwo or more thereof, and a, b and c are such that the compound isstoichiometric, the particle having a size less than 100 nm.
 2. Aparticle according to claim 1 wherein X is Eu, Dy, Tb, Ce, Sm, Er, Gd,Th or Pr.
 3. A particle according to claim 1 wherein X is magnesium,calcium, zinc, bismuth, tin or lead.
 4. A particle according to claim 1wherein Y is tungsten, vanadium, molybdenum, niobium or tantalum.
 5. Aparticle according to claim 4 wherein Y is tungsten.
 6. A particleaccording to claim 1 which has a size from 1 to 50 nm.
 7. A particleaccording to claim 6 which has a size from 2 nm to 10 nm.
 8. A particleaccording to claim 1 of europium tungstate, terbium tungstate, europiummolybdenate, or a mixed vanadate/tungstate salt of gadolinium andeuropium.
 9. A particle according to claim 1 which is coated with anorganic acid or Lewis base.
 10. A particle according to claim 9 which iscoated with a Lewis base.
 11. A particle according to claim 10 which iscoated with a phosphate.
 12. A particle according to claim 11 which iscoated with sodium hexametaphosphate.
 13. A particle according to claim1 which is substantially crystalline.
 14. A process for preparing aparticle of a compound of the formula: X_(a)(YO_(b))_(c) wherein X is arare earth metal or a metal of Group IIA, IIB, IVB or VB of the PeriodicTable, or a mixture of two or more thereof, Y is a metal which forms ananion with oxygen, or a mixture of two or more thereof, and a, b and care such that the compound is stoichiometric, the particle having a sizeless than 100 nm which comprises mixing an aqueous solution having abasic pH of a compound containing an anion of Y and a surfactant whichis an organic acid of Lewis base, with an aqueous solution of a compoundcontaining the cation X.
 15. A process according to claim 14 wherein awater soluble compound containing an anion of Y is added to water, thesurfactant is added to it, the pH is increased, if desired, and asolution of a water soluble compound containing the cation X is added.16. A process according to claim 15 wherein the pH is adjusted to atleast
 8. 17. A process according to claim 14 wherein the cation Y isadded as an alkali metal salt.
 18. A process according to claim 14wherein the water soluble salt of X is a halide.
 19. A process accordingto claim 14 wherein the surfactant is a phosphate, polyvinylpyrrolidoneor an vinyl carboxylic polymer.
 20. A process according to claim 19wherein the surfactant is sodium hexa-meta-phosphate or a copolymer ofacrylic acid and vinyl phosphonate
 21. A process according claim 14wherein the particles are precipitated by the addition of a non-aqueoussolvent.
 22. A process according to claim 14 wherein the surfactant issuch as will provide a surface reactive group.
 23. A process accordingto claim 22 wherein the group is —OH, —SH, —COOH or —N⁺.
 24. (Canceled).25. A particle prepared by a process as claimed in claim
 14. 26. Asecurity marking ink which comprises a particle as claimed in claim 1together with an aqueous or non-aqueous solvent and a binder.
 27. An inkaccording to claim 26 wherein the binder is a carboxylated acrylic resinor an ethylene/vinyl ester copolymer.
 28. A biotag which comprises aparticle as claimed in claim 9 which is attached to one member of aligand binding pair.
 29. A biotag according to claim 28 wherein thebinding pair is avidin and biotin or an antibody and an antigen. 30.(Canceled).
 31. A process for tagging a moiety which comprises attachinga biotag as claimed in claim 28 either directly or after attaching tosaid moiety the other member of said ligand binding pair.
 32. A processaccording to claim 31 wherein the biotag is produced with the aid of across linking agent.
 33. (Canceled).